1. Field of the Invention
The present invention relates to a scanning probe microscope incorporating an optical microscope, and more particularly, to a scanning probe microscope with an optical microscope capable of providing visible images of optically transparent specimens.
2. Description of the Related Art
A scanning probe microscope is an apparatus for measuring configurations and the like of a specimen with resolutions on the order of sub-nanometers on the basis of an interaction between the specimen and a probe. An example of an atomic force microscope is described in U.S. Pat. No. 4,724,318. The atomic force microscope is a typical scanning probe microscope, which measures a force acting between the probe supported on an elastic body and the specimen as a displacement of the elastic body, and determines the configurations of the specimen on the basis of this displacement.
Generally, in a scanning probe microscope, horizontal scanning and vertical position control are carried out by using a tube actuator or so-called tube scanner.
Also known is a scanning probe microscope that incorporates an optical microscope for positioning between the specimen and the probe. The optical microscope, which can provide an image that serves as a control, is very useful to the evaluation of scanning probe microscope (SPM) measurement results, as well as to the positioning of the scanning probe microscope. In general, an optical microscope, such as the one described in International Publication No. WO 89/01603, is incorporated in a manner such that its optical axis is in line with the probe axis.
Objects to be observed by means of conventional scanning probe microscopes were formerly limited to opaque substances, such as metals, semiconductors, etc. Recently, however, there has been an increasing demand for observations of biological specimens using these microscopes.
Generally, biological specimens have high optical transmissions, so that transparent illumination is employed. The biological specimens are observed by using a kind of special optical microscopes, since conventional optical microscopes cannot provide high-contrast optical images due to the high optical transmissions of the specimens. Special optical microscopes include phase-contrast microscopes, modulation-contrast microscopes, and differential interference microscopes. These special microscopes can visualize optically transparent specimens.
FIG. 19 shows an example of a scanning probe microscope that incorporates a special optical microscope. As shown in FIG. 19, the scanning probe microscope comprises an inverted microscopic optical system and a transparent illumination optical system, which constitute a phase-contrast microscope in cooperation with each other.
An illumination optical system 10 is supported by an arm 6, which is attached to the upper end of a supporting column 4 on a base 2. The optical system 10 includes a light source 12, a collector lens 14, a slider 16, and a condenser lens 18. The slider 16, which is movable across an optical path, has a ring slit 16a for phase-contrast microscopy, which selectively transmits a part of illumination light from the light source 12, and a light transmitting section 16b, which transmits the light from the light source 12. If necessary, the slit 16a is situated in the optical path.
A Z-stage 20 is attached to the supporting column 4. The Z-stage 20 includes a stationary section 20a fixed to the column 4 and a moving section 20b movably supported by the stationary section 20a. An arm 22 is fixed to the movable section 20b, and retains the upper end portion of a piezoelectric tube scanner 24. A head 30 is held on the lower end of the scanner 24. A cantilever chip 40 is attached to the underside of the head 30. The chip 40 has a cantilever extending from a supporting portion, and a projection or probe is formed on the underside of the distal end of the cantilever. The head 30 has therein a displacement measurement optical system for measuring the displacement of the cantilever of the cantilever chip 40. This measurement optical system includes a laser diode 32, a focusing lens 34, reflecting means 36, and a photodiode 38 having a plurality of light receiving portions. The displacement of the cantilever of the cantilever chip 40 is obtained on the basis of the position of incidence of reflected laser beam from the cantilever upon the photodiode 38, that is, differences between outputs from the light receiving portions.
A slide glass 50, which carries a biological specimen thereon, is placed on a specimen stage 52 that is fixed to the supporting column 4. An objective lens 58 for phase-contrast microscopy is located under the stage 52 and fixed to a retaining member 54 that is set up on the base 2. Reflecting means 64 is located under the objective lens 58, and a CCD camera 66 for microscopic image observation is provided in the path of reflection of the reflecting means 64.
The working distance of the condenser lens 18 is 182 mm, the piezoelectric tube scanner 24 has an inside diameter of 28 mm and a length of 80 mm, and the head 30 is 20 mm high. Thus, the numerical aperture of the upper end of the scanner 24 is 0.14 (=14/(14.sup.2 +100.sup.2).sup.1/2). The condenser lens 18 has a numerical aperture of 0.1, and illumination light from the lens 18 cannot be intercepted by the scanner 24. Objective lenses having powers of 4.times., 10.times., and 20.times. magnifications are applicable to this illumination.
Since the illumination light for irradiating the specimen is restricted by the top aperture of the piezoelectric tube scanner 24, the numerical aperture of the illumination optical system 10 cannot be larger than 0.14 even though the condenser lens 18 has a large numerical aperture. The optical microscope provides a resolution of 940 nm for an objective lens with a numerical aperture of 0.6 and illumination light with a wavelength of 570 nm.
Applicable objective lenses are allowed to have a power of 20.times. magnification at the most, and objective lenses of 40.times. magnification, which are frequently used in phase-contrast microscopy, cannot be employed for the purpose. This is because the objective lenses of 40.times. magnification have numerical apertures of 0.55 to 0.6 such that the numerical apertures of ring slits corresponding to these lenses are larger than that of the upper end of the piezoelectric tube scanner.